PROJECT SUMMARY OF THE FUNDED AWARD Heparin-related glycosaminoglycans (GAGs) hold enormous promise in the field of regenerative medicine, and also are a largely unexploited source of therapeutic agents to address a range of other medically important conditions including cancer and neurodegenerative diseases. Nevertheless, their practical utilization in the clinic is still confined to anticoagulants, while progress in other areas remains relatively modest. The great challenge presented by these biopolymers to medicine and, more broadly, to structural biology is due in large part to their enormous structural heterogeneity, which is mirrored by their multiple roles in modulating angiogenesis, cell adhesion, embryogenesis, inflammation, metastasis and wound healing. Despite extensive research efforts, clear understanding of how various structural features modulate function of these biopolymers remains wanting, arguably because the inquiry was designed using the framework of the lock-and-key model, a paradigm that had been tremendously successful in structural biology applied to proteins. However, this approach fails to recognize that the dramatically higher level of structural diversity exhibited by GAGs has functional importance. The proposed research seeks to shift the prevailing paradigm in the field of GAG/protein interactions by moving away from the notion of well-defined sterically complementary binding sites and emphasizing a greater role of polyvalent electrostatics. In addition to revealing the determinants of GAG/protein interactions and understanding how they fine-tune the binding process, we will obtain a high-resolution and dynamic description of the mechanism of heparin interaction with several therapeutically relevant proteins. This will be done without either limiting the structural space to a few precisely defined GAG molecules or reducing the conformational space to a few static snapshots available from crystal structures. Towards this goal, we will adopt a ?holistic? approach to protein/GAG interactions that embraces the structural diversity of heparin-like GAGs, as opposed to the commonly accepted approach which limits the scope of inquiry to a few molecules accessible through synthesis. We will use a combination of bottom-up and top-down approaches to study protein/GAG interactions, the former focusing on relatively well-defined subsets of short heparin oligomers obtained by affinity separations, and the latter aimed at intact heparin. This will reveal the structural properties governing the interactions of GAGs with several therapeutically relevant proteins. Novel applications of gas-phase ion chemistry (electron capture and collisional activation) will be developed for characterization of large heterogeneous GAG/protein complexes. Finally, atomic-level Monte Carlo simulations in parallel with H/D exchange studies will lead to a high-resolution and dynamic depiction of the interaction process. This new view of GAG/protein interactions will allow the therapeutic potential of heparin-like GAGs to be exploited more efficiently in areas as diverse as regenerative medicine, inflammation, neurodegenerative disorders and oncology.